SWISS-MODEL Workspace
Create a user account for SWISS-MODEL Workspace
http://swissmodel.expasy.org/workspace/
1. SWISS-MODEL Repository
1. Retrieve a precomputed homology model for the platypus myoglobin
MYG_ORNAN from the SWISS-PROT Repository.
Q1.1 What template structure has been used in the modeling process?
Pig myoglobin (1myh.pdb)
Q1.2 What is the sequence identity percentage between the target and the
template?
Sequence identity:
84%
Q1.3 Is this sufficient for reliable homology modeling?
Yes. Target/template sequence identity above 50 % will typically yield high
quality homology models.
2. Download the model as a Swiss-PDB Viewer project file and open the file
in DeepView.
Identify the residues that are different between the target and the
template.
Hint: Select variable positions
Select > Residues Identical to Reference Layer
Select > invert selection
3. Create a visualisation that allows you to see the side chains in the variable
positions relative to the overall fold of the protein. Copy paste an image of
your visualization below.
Q1.4 Are any of the variable residues located in the hydrophobic core of the
protein?
None of the variable residues are located in the hydrophobic core. All of them are
on the surface, which typically evolves faster than the core.
2. Alignment mode & comparing models
2.1 Automated mode
Use sequence_11B from sequence assignments list as TARGET
Submit TARGET sequence to Swissmodel's automatic mode
- note sequence identity, e-values from template search
- look at the quality profiles
- download model, send to SolvX server (must check 'Yes' first chain only)
3.2. Swissmodel alignment mode with manually improved GTG alignment
The TARGET was submitted to GTG server. The best template identified from
GTG results is 1j6oA. Below is the GTG's alignment in clustal W format:
CLUSTAL W
QUERY YSDIHLHYVDFFQ----ESAGITKLL---EGVASAKIEHAVFTGISVAKEWDENEPVQPRYYAG-DDAKAYW----YSAT
1J6O-A XVDTHAH-LHFHQFDDDR------NAVISSFEENNIEFVVNV---------------G-VNL-EDSKKSLDLSKTSDR-QUERY DILIAHAYKQLPTEAKTRFHPFISGFNPNDMNAATQIKNMLDRDPGLWQGIGEVFTRHDYLTALIYGKAPRANSRALAKV
1J6O-A -------IFCSVGVH-----PH---DAKEVPEDFIEHLEKFAKDEKVVAIGETGLDFFRNISPAEVQKRVFVEQIELAGK
QUERY YRVAAEYDLPVLLHSNITSKRERSPLYLGEFEEALRANPDVRFIWSHAGTSMEIYRSQGKMAFLLPTVEQLLGKYPNLYI
1J6O-A LNLPLVV------HIRDAYSEAYEILRTES-------LPEKRGVIHAFSSDYE----------WA-K--KFIDLGFLLGI
QUERY DLSWSVLRPYLVDELGNP-DPAWLDLVSRYPTRFMLGSD
1J6O-A GG------PVTYPKNEALRE-----VVKRVGLEYIVLET
Manually refined alignment
Swissmodel in Alignment mode could not build a model because loops did not
close. Liisa has manually edited the alignment, moving inserts and gaps to
suitable positions (turns), adding C-terminal sequence to the alignment, and
doing five cycles of refinements to the alignment in order optimize the SolvX
profile. This is the alignment after five attempts (in fasta format):
>1j6oA
HHHHXVDTHAHLH-FHQFDDDRNAVISSFEENNIEFVVNV---------------GVNLEDSKKSLDLSKT-SDR
IFCSVGVHPHDAKEV------------PEDFIEHLEKFAKDEKVVAIGETGLDFFRNIS------PAEVQKRVFVEQ
IELAGKLNLPLVVHIRDAYSEAYEILRTES--------LPEKRGVIHAFS------------SDYEWAKKFIDLGFLLGI
GGPVTYP----------KNEALREVVKRVGLEYIVLET
DCPFLPPQPFRGKRNEPKYLKYVVETISQVLGVPEAKVDEATTENARRIFLEVKE
>TARGET_5
----YSDIHLHYVDFFQESAGITKLLEGVASAKIEHAVFTGISVAKEWDENEPVQPRYYAGDDAKAYWYSATDIL
IAHAYKQLPTEAKTRFHPFISGFNPNDMNAATQIKNMLDRDPGLWQGIGEVFTRHDYLTALIYGKAPRANSRALAKV
YRVAAEYDLPVLLHSNITSKRERSPLYLGEFEEALRANPDVRFIWSHAGTSMEIYRSQGKMAFLLPTVEQLLGKYPNLYI
DLSWSVLRPYLVDELGNPDPAWLDLVSRYP-TRFMLGS
DVVGTFGNLSKYMFGFEPFLDALPHDIAHRVALSNLISILPKARQKEALDMG---
Send this alignment to Swissmodel in Alignment mode. Download the model and
send to SolvX server.
Compare the models from Automated mode and manually edited alignment
submitted to Alignment mode. Look at the global quality scores and the quality
profiles.
Q2.1 - Which of these two models has better quality?
Manually edited alignment
Automated mode: solvx -29.3
Manual mode: solvx -44.5
Q2.2 - What are the SolvX scores for these two models? Which one is better?
The Manually edited alignment
Lessons: getting the alignment right is difficult. Manually editing the alignment
can improve the quality of a 3D model. It is difficult to achieve net negative, not
to mention native-like, SolvX profiles if the template is wrong.
Swissmodel uses two templates, which are structurally similar to 1j6oA.
Swissmodel's alignments are very bad though the templates are correct.
3 Phyre server
Submit sequence_11B to the Phyre modelling server at
http://www.sbg.bio.ic.ac.uk/phyre/
Analyse the resulting model using SolvX-server.
Compare the SolvX score of the model generated by Phyre to those created using
Swissmodel server.
Q3.1 - What is the solvation score of the Phyre model?
-143.9
Models generated by the Phyre server appear to have varying solvation
scores.
Q3.2 - Based on the SolvX score which server gives better results for this
sequence?
Phyre: -143.9
Swissmodel alignment mode: -50.7
Phyre is gives better results in this case
4 SWISS-MODEL Alignment mode
Sequence Alignment is the most important step in homology modeling. Incorrect
alignment will lead to wrong 3D model. Alignment between two sequences can
be obtained by pairwise alignment method (e.g. BLAST). However, in most cases
alignment can be improved using multiple sequence alignment.
Model the structure of the protease sequence (target) below:
>Murine_HV
SGIVKMVSPTSKVEPCIVSVTYGNMTLNGLWLDDKVYCPRHVICSSADMTDPDYPNLLCRVTSSDFCVMSGRMSLTVMSYQMQGCQ
LVLTVTLQNPNTPKYSFGVVKPGETFTVLAAYNGRPQGAFHVTLRSSHTIKGSFLCGSCGSVGYVLTGDSVRFVYMHQLELSTGCH
TGTDFSGNFYGPYRDAQVVQLPVQDYTQTVNVVAWLYAAIFNRCNWFVQSDSCSLEEFNVWAMTNGFSSIKADLVLDALASMTGVT
VEQVLAAIKRLHSGFQGKQILGSCVLEDELTPSDVYQQLAGVKLQ
1. Identify structural templates from the exPDB database for your target
protein by using template search at :
SWISS-Model Workspace > Tools > Template Identification
To make the search faster check Gapped Blast Query only!
Q4.1 What is the highest scoring template?
3d23 (A, B, C or D chain)
2. The file proteases.fasta contains sequences of homologous Corona virus
proteases.
Retrieve the sequence for the highest scoring template pdb-file, and add it
to the file containing protease family sequences.
3. Align the sequences using Muscle multiple sequence alignment program.
Inspect your alignment in Jalview for any alignment problems.
4. Save your alignment in fasta format
5. Upload your alignment to:
SWISS-MODEL Workspace > Alignment Mode
Note: You may need to edit the template sequence name in order for the server
to be able to parse the alignment information correctly.
6. Download your model as a Swiss-PDB Viewer project file and open it in
DeepView
7. Color the target structure by Protein Problems
Protein Problems colour scheme:
This menu will affect the color of the backbone and of the sidechain separately
irrespective of the 'Color act on...' current setting. Residues with peptide bonds
too long will be colored in pink, while the rest of the protein is colored in grey.
This is very useful during modeling. In addition, the backbone of residues with
phi/psi angles laying in "forbidden" zones will be colored in yellow (except
GLY that are ignored) and Prolines whose backbone will be colored in red. Also,
the sidechains of residues capable of H-bonding that are not participating
in any H-bond with other residues are colored in orange. Finally, Clashes will
also be computed and displayed in pink dotted lines. Residues with clashes will
be selected.
http://spdbv.vital-it.ch/color_menu.html
Q 4.2 Which residue is flagged as lacking expected hydrogen bonds (orange)?
Trp31, Cys44, Asp48, Met49, Asn97, His134, Trp207, Cys225, Glu260, Gln277
(Orange)
Q 4.3 Which residues are flagged as having forbidden main chain bond angles?
Leu27, Asp33, Gln221 (yellow)
Leu27, Pro184 (red)
8. Look at the ANOLEA and GROMOS plots in SWISS-MODEL workspace
Q 4.3 Are there any residues with high scores (high energy) in the model?
No there are no high scoring residues with is a good thing.
9. Generate solvation profiles for your model and your template using Solvxserver. Copy paste the profiles below.
Q 4.4 What are the overall solvation scores for the target and the template?
Target: Overall Score: -68.4
Template Structure: 3D23-D , Overall Score: -72.0